1. Protein Purification and Characterization Techniques
Nafith Abu Tarboush, DDS, MSc, PhD

2. Extracting Pure Proteins from Cells
Purification techniques focus mainly on size & charge
The first step is homogenization (grinding, Potter–Elvejhem homogenizer, sonication, freezing and thawing, detergents)
Differential centrifugation (600 g: unbroken cells & nuclei; 15,000 g: mitochondria; 100,000 g: ribosomes and membrane fragments)

3. Salting in & out Are proteins soluble? If yes, to which limit?
Salt stabilizes the various charged groups on a protein molecule and enhance the polarity of water, thus attracting protein into the solution and enhancing the solubility of protein
Ammonium sulfate is the most common reagent to use at this step
This technique is important but results are crude

4. Dialysis
Principle of diffusion
Concept of MW cut-off
Pure vs. crude

5. Column Chromatography
Greek chroma, “color,” and graphein, “to write”
Is it just for colourful proteins?
Chromatography is based on two phases: stationary & mobile
What are the different kinds?

6. Size-exclusion chromatography Gel-filtration chromatography
Separation on the basis of size (MW)
Stationary (cross-linked gel particles): consist of one of two kinds of polymers; the 1st is a carb. polymer (ex. dextran or agarose); often referred to by Sephadex and Sepharose. The 2nd is based on polyacrylamide (Bio-Gel)
Extent of crosslinking & pore size (exclusion limit)
Convenient & MW estimate
Each gel has range of sizes that separate linearly with the log of the molecular weight

7. Molecular-sieve chromatography

8. Affinity chromatography
It has specific binding properties
The polymer (stationary) is covalently linked to a ligand that binds specifically to the desired protein
The bound protein can be eluted by adding high conc. of the soluble ligand
Protein–ligand interaction can also be disrupted with a change in pH or ionic strength
Convenient & products are very pure (Antigen-antibody, His-tag, GST-Tag)

9. Ion-exchange chromatography
Interaction based on net charge & is less specific
Resin is either negatively charged (cation exchanger) or positively charged (anion exchanger)
Buffer equilibration, exchange resin is bound to counter-ions. A cation-exchange resin is usually bound to Na+ or K+ ions, and an anion exchanger is usually bound to Cl– ions
Proteins mixture loading
Elution (higher salt concentration)

10. Electrophoresis
Based on the motion of charged particles in an electric field
Macromolecules have differing mobilities based on their charge, shape, and size
The most common medium is a polymer of agarose or acrylamide

11. Agarose vs. PAGE Agarose (nucleic acids), PAGE (proteins)
In PAGE: SDS or NO-SDS {CH3(CH2)10CH2OSO3Na+}
SDS completely denatures proteins (multi-subunit proteins)
Acrylamide offers higher resistance to large molecules
Shape and charge are approximately the same (sizes is the determining factor)
Acrylamide without the SDS (native gel): study proteins in their native conformation (mobility is not an indication of size)

12. Isoelectric focusing Proteins have different isoelectric points
Gel prepared with a pH gradient parallel to electric-field gradient
Two-dimensional gel electrophoresis (2-D gels)

13. Immunoassays – Western blot
From gel to a membrane (nitrocellulose or polyvinylidene difluoride, PVDF)
Detection:
Colorimetric: enzymes bound to 2nd Ab
Chemiluminescent: reporter 2nd Ab
Radioactive detection: X-rays
Fluorescent detection: fluorescently labeled probe

14. Immunoassays - ELISA Enzyme-Linked Immunosorbent Assay
Detect & quantify substances ( peptides, proteins, antibodies & hormones)
Usually done in 96-well polystyrene plates (passively bind antibodies and proteins)
Apllication:
Screening (HIV, Hepatitis B&C)
Hormones (HCG, LH, TSH, T3, T4)
(Green, positive)
(No color, negative)

15. Protein sequencing - Edman Method
Step 1: how much and which amino acids are involved
Hydrolysis (heating + HCl) & Separation (ion-exchange chromatography or by high performance liquid chromatography, HPLC)

16. Protein sequencing - Edman Method
Step 2: determining the identities of N-terminal and C-terminal ends of protein
Necessary esp. to determine if the protein consists of one or two polypeptide chains
Steps 3: cleavage into smaller fragments (Edman degradation)
Enzymes- Trypsin, Chymotrypsin
Chemical reagents- Cyanogen bromide CNBr

17. Trypsin: Cleaves @ C-terminal of (+) charged side chains
Chymotrypsin: C-terminal of aromatics
CNBr: C-terminal of INTERNAL methionines

18. Protein sequencing – Mass Spectrometry
Mass/charge ratio

19. Protein sequencing – prediction from DNA & RNA
If the sequence of the gene is known, this is very easy
If the sequence of the gene is unknown (newly isolated proteins)? Sequence a short segment, complementary RNA, isolate mRNA, PCR, gene sequencing